Because of their relatively small number of neurons and simple neural circuitry, invertebrate preparations are advantageous for studying the relationship between cellular events and behavior. We propose to capitalize on this advantage by exploring the relationships between the behavioral, cellular and molecular levels during dishabituation, sensitization, and classical conditioning of the gill-withdrawal reflex in Aplysia. Previous studies supported by this grant have suggested that a mechanism of dishabituation and sensitization of the reflex is presynaptic facilitation of the siphon sensory neurons, due in part to cAMP-dependent protein phosphorylation, and that a mechanism of conditioning of the reflex is activity dependent amplification of the facilitation. However, the studies performed at the behavioral, cellular, and molecular levels have generally used different preparations and procedures, so that the relationship between the different levels has been inferred rather than tested directly. Moreover, there is now evidence that multiple cellular and molecular mechanisms may contribute to each of these forms of learning, so that the contribution of each mechanism is uncertain. In the past several years, we have developed two preparations with which it is possible to bridge the levels of analysis and examine the contribution of events at one level to events at a higher level. In this grant renewal, we propose to utilize these preparations to test more rigorously the roles of the mechanisms we have already described, and also to examine additional sites and mechanisms of plasticity that may contribute to dishabituation, sensitization, classical conditioning, and higher-order features of conditioning of the reflex. We will conduct four sets of experiments: (1) Behavioral and cellular studies of dishabituation and sensitization of the gill-withdrawal reflex in an isolated mantle organ preparation; (2) Behavioral and cellular studies of classical conditioning of the reflex; (3) Behavioral and cellular studies of higher-order features of conditioning and (4) Cellular and molecular studies of activity-dependent facilitation in a sensory neuron cluster preparation.